Abstract
Human dopamine D2 (hD2) and D3 (hD3) receptors were expressed at similar, high expression levels in Chinese hamster ovary (CHO) cells, and their coupling to G proteins and further signal transduction pathways were compared. In competition radioligand-binding experiments, guanosine-5′-O-(3-thio)triphosphate (GTPγS) treatment of hD2S- or hD3-CHO cell membranes induced a rightward shift and steeping of the dopamine inhibition curve. This effect was pronounced for hD2 receptors and small for hD3 receptors. Activation of G proteins was investigated in [35S]GTPγS-binding assays. Dopamine stimulated [35S]GTPγS binding 330 and 70% over basal levels on hD2-CHO and hD3-CHO cell membranes, respectively. (+)-7-(Dipropylamino)-5,6,7,8-tetrahydro-2-naphthalenol and PD128907 were partial agonists for both receptors. Haloperidol, risperidone, raclopride, and nemonapride inhibited dopamine-stimulated [35S]GTPγS binding with potencies comparable to their binding affinities for hD2 and hD3 receptors in CHO cell membranes; inverse agonism could not be detected with this assay. Receptor stimulation by dopamine inhibited forskolin-induced cyclic AMP formation in hD2-CHO and hD3-CHO cells by 70%. Furthermore, the extracellular acidification rate increased when hD2-CHO and hD3-CHO cells were stimulated by dopamine; this effect was abolished by pertussis toxin pretreatment. In this study, we could demonstrate clear functional effects at different levels of the signaling cascade of hD2and hD3 receptors in CHO cells when expressed at high levels. High-affinity agonist binding to hD2 and hD3 receptors was still present, but effects of receptor-G protein uncoupling at hD3 receptors were small, indicating that hD3 receptors maintain relatively high-affinity agonist binding in the absence of G proteins.
Footnotes
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Send reprint requests to: Dr. Josée E. Leysen, Department of Biochemical Pharmacology, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium. E-mail:jleysen2{at}janbe.jnj.com
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1 This work was supported by a grant from the IWT (Vlaams Instituut voor de Bevordering van het Wetenschappelijk-Technologisch Onderzoek in de Industrie); project IWT 940232.
- Abbreviations:
- human dopamine D2
- cAMP, cyclic AMP
- CHO
- Chinese hamster ovary
- CSS
- controlled salt solution
- DMEM
- Dulbecco’s modified Eagle’s medium
- DMSO
- dimethyl sulfoxide
- hD2L-CHO
- CHO cells stably expressing cloned human D2L receptors
- hD3-CHO
- CHO cells stably expressing cloned human D3 receptors
- cIC50
- corrected IC50
- [35S]GTPγS
- [35S]guanosine-5′-O-(3-thio)triphosphate
- 7-OH-DPAT
- (+)-7-(dipropylamino)-5,6,7,8-tetrahydro-2-naphthalenol
- hD2
- hD3, human dopamine D3
- FCS
- fetal calf serum
- pIC50 = −log IC50
- IC50, concentration of the compound producing 50% inhibition of the specific binding of the radioactive ligand)
- pEC50 = −log EC50
- EC50, concentration of the compound producing 50% effect)
- Received February 1, 1999.
- Accepted March 26, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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